Identification of nucleotides critical for activity of the sigmaE-dependent rpoEp3 promoter in Salmonella enterica serovar Typhimurium

We previously described a two-plasmid system for the identification of promoters recognized by Salmonella enterica serovar Typhimurium (S. Typhimurium) sigmaE. The S. Typhimurium sigmaE-dependent rpoEp3 promoter was active in the E. coli two-plasmid system only after arabinose-induced expression of S. Typhimurium rpoE. In the present study, we have exploited this two-plasmid system for the identification of nucleotides critical for activity of the rpoEp3 promoter. A library of randomly mutated DNA fragments containing the rpoEp3 promoter was cloned upstream of a lacZalpha reporter gene and screened for activity in the presence of S. Typhimurium sigmaE. The clones exhibiting reduced LacZ activity were sequenced to identify the mutations. The activity of the mutated rpoEp3 promoters were studied further using a luciferase-based promoter-probe plasmid. All of the important nucleotides of the rpoEp3 promoter (in capital) were located in the -35 (ggAActt) and -10 (TctaA) regions. The critical nucleotides were also the most conserved in known sigmaE-dependent promoters. The study also revealed the importance of the 16-bp spacing between -10 and -35 region, as reducing the spacing to 15-bp greatly reduced activity of the promoter. This method should be generally applicable for the identification of important nucleotides in the cognate promoters of other sigma factors.     

Amplification of the groESL operon in Pseudomonas putida increases siderophore gene promoter activity

Molecular Genetics and Genomics - Pseudobactin 358 is the yellow-green fluorescent siderophore [microbial iron(III) transport agent] produced by Pseudomonas putida WCS358 under iron-limiting...     

Incidence and Identification of Pseudomonas fluorescens and Pseudomonas putida in the Clinical Laboratory

Strains of Pseudomonas producing fluorescin but no pyocyanin or pyorubrin were studied by biochemical and antibiotic sensitivity testing. A rapid nitrate test was found to be useful in distinguishing P. aeruginosa (positive) from P. fluorescens and P. putida (both negative). A shortened gelatin test differentiated P. fluorescens (positive) from P. putida (negative). P. fluorescens and P. putida were very sensitive to low levels of kanamycin and resistant to carbenicillin, a pattern just the opposite of that obtained with P. aeruginosa.     

Differential DNA bending introduced by the Pseudomonas putida LysR-type regulator, CatR, at the plasmid-borne pheBA and chromosomal catBC promoters

The plasmid-borne pheBA operon of Pseudomonas putida strain PaW85 allows growth of the host cells on phenol. The promoter of this operon is activated by the chromosomally encoded LysR-type regulator CatR, in the presence of the inducer cis, cis-muconate. cis, cis -muconate is an intermediate of catechol degradation by the chromosomally encoded ortho or β-ketoadipate pathway. The catBC operon encodes two enzymes of the β-ketoadipate pathway and also requires CatR and cis, cis-muconate for its expression. The promoters of the pheBA and catBC operons are highly homologous, and since both respond to CatR, it is likely that the pheBA promoter was recruited from the ancestral catBC promoter. Gel shift assays and DNase I footprinting have shown that the pheBA promoter has a higher binding affinity for CatR than the catBC promoter. Like the catBC promoter, the pheBA promoter forms two complexes (C1 and C2) with CatR in the absence of cis, cis-muconate, but only forms a single complex (C2) in the presence of cis, cis-muconate. Like the catBC promoter CatR repression binding site (RBS) and activation binding site (ABS) arrangement, the pheBA promoter demonstrates the presence of a 26 bp segment highly homologous to the RBS that is protected by CatR from DNase I digestion in the absence of the inducer. An additional 16 bp sequence, similar to the catBC promoter ABS, is protected only when the inducer cis-cis-muconate is present. The binding of CatR in absence of cis, cis -muconate bends the catBC and pheBA promoter regions to significantly different degrees, but CatR binding in the presence of cis, cis-muconate results in a similar degree of DNA bending. The evolutionary implications of the interactions of CatR with these two promoters are discussed.     

Sigma 54 levels and physiological control of the Pseudomonas putida Pu promoter

The cellular levels of the alternative sigma factor sigma(54) of Pseudomonas putida have been examined in a variety of growth stages and culture conditions with a single-chain Fv antibody tailored for detection of scarce proteins. The levels of sigma(54) were also monitored in P. putida strains with knockout mutations in ptsO or ptsN, known to be required for the C-source control of the sigma(54)-dependent Pu promoter of the TOL plasmid. Our results show that approximately 80 +/- 26 molecules of sigma(54) exist per cell. Unlike that in relatives of Pseudomonas (e.g., Caulobacter), where fluctuations of sigma(54) determine adaptation and differentiation when cells face starvation, sigma(54) in P. putida remains unexpectedly constant at different growth stages, in nitrogen starvation and C-source repression conditions, and in the ptsO and ptsN mutant strains analyzed. The number of sigma(54) molecules per cell in P. putida is barely above the predicted number of sigma(54)-dependent promoters. These figures impose a framework on the mechanism by which Pu (and other sigma(54)-dependent systems) may become amenable to physiological control.     

Hydantoinase activity of immobilized non-growing Pseudomonas putida cells

Summary Hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) activity of Pseudomonas putida DSM 84 was evaluated using cells immobilized in alginate beads and in a microporous hollow fibre bioreactor. Conversion of dihydrouracil into N-carbamyl--alanine was most efficient with alginate-immobilized cells. A 40 to 45% conversion was obtained in shake flasks and in continuous mode with packed bed columns. The highest volumetric productivity was obtained with a packed bed column operated at a dilution rate of 0.5 h^-1 (99 g of product. 100 l^-1 per hour). After 96 h the alginate beads began to swell and break apart; no free cells were detected however. Despite some initial loss of cells from the microporous hollow fibre bioreactor, a steady state was later established and maintained for 400 h at dilution rates of 0.1 and 0.25 h^-1.     

Caffeine-inducible enzyme activity in Pseudomonas putida ATCC 700097

Caffeine (1,3,7-trimethylxanthine), a ubiquitous component of human diet has been suggested as a chemical indicator of ecosystem impacts of sewage spills and treated effluent discharges because it is not sufficiently metabolized by wastewater microorganisms. This study identified enzymes responsible for caffeine metabolism in sewage bacteria. Pseudomonas putida biotype A (ATCC 700097) originally isolated as a rare caffeine-degrading organism in domestic wastewater exhibited diauxic growth on caffeine, concomitant with the expression of a P450-type cytochrome and peroxidase enzyme activities. Initial growth phase lasted 13.8 ± 1.4 h with a growth rate that was five times slower than the secondary growth phase that lasted 5.5 ± 1.2 h. Molecular and enzymatic characteristics of the cytochrome P450-type enzyme differ from the previously described cytochrome P450 (P450_cam) of P. putida (ATCC 17453) involved in camphor metabolism. The caffeine-inducible cytochrome P450-type enzyme exhibited a carbon monoxide difference spectrum peak at 450 nm, but does not allow growth on camphor. Caffeine induced production of haem-associated peroxidase activity was confirmed with 3,3, 5,5-tetramethylbenzidine–H₂O₂ reaction in polyacrylamide gels. Polymerase chain reaction (PCR) primers derived from the gene for cytochrome P450_cam (camC) of P. putida (ATCC 17453) did not yield an amplification product when DNA extracted from P. putida strain ATCC 700097 was used as template. The data demonstrate that caffeine is metabolized through a specific biphasic pathway driven by oxygen-demanding enzymes.     

Mutational analysis of the influenza virus cRNA promoter and identification of nucleotides critical for replication

Replication of the influenza A virus virion RNA (vRNA) requires the synthesis of full-length cRNA, which in turn is used as a template for the synthesis of more vRNA. A "corkscrew" secondary-structure model of the cRNA promoter has been proposed recently. However the data in support of that model were indirect, since they were derived from measurement, by use of a chloramphenicol acetyltransferase (CAT) reporter in 293T cells, of mRNA levels from a modified cRNA promoter rather than the authentic cRNA promoter found in influenza A viruses. Here we measured steady-state cRNA and vRNA levels from a CAT reporter in 293T cells, directly measuring the replication of the authentic influenza A virus wild-type cRNA promoter. We found that (i) base pairing between the 5' and 3' ends and (ii) base pairing in the stems of both the 5' and 3' hairpin loops of the cRNA promoter were required for in vivo replication. Moreover, nucleotides in the tetraloop at positions 4, 5, and 7 and nucleotides forming the 2-9 base pair of the 3' hairpin loop were crucial for promoter activity in vivo. However, the 3' hairpin loop was not required for polymerase binding in vitro. Overall, our results suggest that the corkscrew secondary-structure model is required for authentic cRNA promoter activity in vivo, although the precise role of the 3' hairpin loop remains unknown.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.     

Roles of CatR and cis,cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida.

In Pseudomonas putida, the catBC operon encodes enzymes involved in benzoate degradation. Previous studies have determined that these enzymes are induced when P. putida is grown in the presence of benzoate. Induction of the enzymes of the catBC operon requires an intermediate of benzoate degradation, cis,cis-muconate, and a regulatory protein, CatR. It has been determined that CatR binds to a 27-bp region of the catBC promoter in the presence or absence of inducer. We have called this the repression binding site. In this study, we used a gel shift assay to demonstrate that the inducer, cis,cis-muconate, increases the affinity of CatR for the catBC promoter region by 20-fold. Furthermore, in the absence of cis,cis-muconate, CatR forms two complexes in the gel shift assay. The inducer cis,cis-muconate confers specificity primarily for the formation of complex 2. DNase I footprinting showed that an additional 27 bp of the catBC promoter region is protected by CatR in the presence of cis,cis-muconate. We have named this second binding site the activation binding site. Methylation interference footprinting determined that in the presence or absence of inducer, five G nucleotides of the catBC promoter region were necessary for CatR interaction with the repression binding site, while a single G residue was important for CatR interaction with the activation binding site in the presence of cis,cis-muconate. Using polymerase chain reaction-generated constructs, we found that the binding of CatR to the repression binding site is independent of the activation binding site. However, binding of CatR to the activation binding site required an intact repression binding site.     

Involvement of IHF protein in expression of the Ps promoter of the Pseudomonas putida TOL plasmid.

Regulation of the xyl gene operons of the Pseudomonas putida TOL plasmid is mediated by the products of the downstream clustered and divergently oriented xylR and xylS regulatory genes. The xylR-xylS intergenic region contains the xylR and xylS promoters Pr and Ps, respectively. A binding site for the XylR activator protein is located upstream of Ps and overlapping Pr. DNase I footprint experiments showed that one of these sites, which overlaps the recognition site for XylR activator, as well as an AT-rich region comprising the Ps promoter consensus were protected by integration host factor (IHF). IHF was found to act negatively in the in vivo activation of the Ps promoter, since the activity of a Ps promoter::lacZ fusion was elevated in an Escherichia coli mutant lacking IHF. In contrast, no alteration in the synthesis of XylR protein in the E. coli IHF-deficient mutant was detected.     

Chemotaxis to Pyrimidines and Identification of a Cytosine Chemoreceptor in Pseudomonas putida

We developed a high-throughput quantitative capillary assay and demonstrated that Pseudomonas putida strains F1 and PRS2000 were attracted to cytosine, but not thymine or uracil. In contrast, Pseudomonas aeruginosa PAO1 was not chemotactic to any pyrimidines. Chemotaxis assays with a mutant strain of F1 in which the putative methyl-accepting chemotaxis protein-encoding gene Pput_0623 was deleted revealed that this gene (designated mcpC) encodes a chemoreceptor for positive chemotaxis to cytosine. P. putida F1 also responded weakly to cytidine, uridine, and thymidine, but these responses were not mediated by mcpC. Complementation of the F1 ΔmcpC mutant XLF004 with the wild-type gene restored chemotaxis to cytosine. In addition, introduction of this gene into P. aeruginosa PAO1 conferred the ability to respond to cytosine. To our knowledge, this is the first report of a chemoreceptor for cytosine.Motile bacteria are capable of detecting chemical gradients in the environment and swim toward or away from them, a behavior known as chemotaxis. Historically, the enteric bacterium Escherichia coli has been the model organism for chemotaxis studies. E. coli has four transmembrane chemoreceptors called methyl-accepting chemotaxis proteins (MCPs), each of which binds a set of chemicals directly or in complex with specific periplasmic binding proteins. MCPs send signals to the flagellar motor via a complex signal transduction system that is composed of six soluble chemotaxis proteins, through which the bacterium modifies its swimming behavior based on the signal(s) received (for reviews, see references 5 and 15). The MCPs of E. coli sense a variety of stimuli, including amino acids, sugars, and dipeptides (30, 44). We recently reported that E. coli also responds to the pyrimidines thymine and uracil and demonstrated that Tap, the MCP known to mediate chemotaxis to dipeptides, is required for pyrimidine taxis (29).Pseudomonads are environmental bacteria that are widespread in nature, and all Pseudomonas species are motile. They have conserved chemotaxis proteins that are homologous to those present in E. coli, but their chemosensory systems appear to be more complex (6, 39, 55). Unlike E. coli, which has only one set of chemotaxis (che) genes in a single gene cluster, Pseudomonas species have multiple che gene homologs organized in several unlinked gene clusters (39). In addition, genome sequence analyses have revealed that Pseudomonas strains have numerous putative MCP genes. For example, the genome of Pseudomonas aeruginosa PAO1 (46) encodes 26 MCP-like proteins, Pseudomonas putida KT2440 (34) has 27, and Pseudomonas syringae DC3000 (9) has 49 (39).The best-studied chemotaxis system in Pseudomonas is that of the opportunistic pathogen P. aeruginosa. More than 75 different chemoattractants have been identified for P. aeruginosa (39), and 13 of its 26 MCP-like proteins have been functionally characterized. Eight MCPs have been shown to mediate positive responses to amino acids (PctABC), inorganic phosphate (CtpH and CtpL), malate (PA2652), ethylene (TlpQ), and chloroethylenes (McpA) (3, 25-27, 42, 47, 54). Two MCPs (McpA and McpB) were shown to be required for general optimal chemotaxis (16), and one MCP-like protein (Aer) was found to mediate energy taxis (22). The MCP-like proteins BdlA and PilJ were shown to be involved in biofilm formation and biosynthesis of type IV pili, respectively (10, 12, 32).P. putida is a common soil bacterium and, unlike P. aeruginosa, is not known to be pathogenic. Although P. putida and P. aeruginosa each have approximately the same number of MCP-like genes in their genomes, most of the protein products show relatively low amino acid sequence similarity. Based on our BLAST searches, three putative P. putida F1 MCPs have no obvious counterparts in P. aeruginosa PAO1. Most of the others share between 30 and 70% amino acid sequence identity, with the highest sequence conservation in the C-terminal signaling domains. The most highly conserved MCP-like proteins in the two species are Aer and PilJ (both are 77% identical to the corresponding homologs). These observations suggest that the two organisms respond to different subsets of attractants, which most likely reflects their different lifestyles and environmental niches. P. putida is known for its catabolic versatility (45), and we expect that members of the species are capable of responding to a correspondingly wide range of organic attractants. We are interested in defining the range of attractant and repellent responses and the functions of the MCPs present in P. putida compared to those of P. aeruginosa. In this study, we used P. putida strains F1 and PRS2000 and P. aeruginosa strain PAO1 to investigate the chemotactic responses to pyrimidines.